Metabolic reprogramming by cancer cells is critical to facilitate their proliferation and survival against micro environmental stresses. Glutamine (Gln) i an essential amino acid that plays a unique role in the metabolism of proliferating cancer cells, providing building blocks to sustain cell proliferation and regulating redox homeostasis and signal transduction pathways. Glutaminase (GLS) is an enzyme that initiates this process by converting glutamine to glutamate which is subsequently used in multiple reactions that support tumor cell growth and survival, including the generation of energy (TCA cycle), synthesis of amino acids and production of glutathione. Importantly, it has also been shown that GLS inhibition can reduce 2HG levels in tumors thus increasing TET activity and leading to decreased methylation. Recent findings from our group and others indicate that AML cells depend on Gln as a major carbon source for growth and survival. Inhibition of glutaminase with novel selective small molecule inhibitor CB-839, or silencing GLS expression with inducible shRNA inhibited mitochondrial respiration, reduced cell growth and induced apoptosis in a subset of leukemia cell lines and primary AML cells. Further, co-culture of leukemia cells with bone marrow-derived mesenchymal stem cells caused metabolic reprogramming in both, stroma and cancer, whereby glycolytic stroma supported oxidative metabolism of leukemic cells, in part by supplying glutamine. Myelodysplastic syndromes (MDS) are incurable malignancies that need newer therapeutic targets. Our preliminary data demonstrate that GLS is overexpressed in AML with complex cytogenetics, in high risk MDS stem cells and is associated with worse prognosis in MDS patients. We hypothesize that the glutaminase is a therapeutic target in MDS and metabolic reprogramming involved in transformation of MDS into AML. We propose to monitor the changes in metabolism in the conversion of MDS to AML in mouse models and determine if inhibiting glutaminolysis inhibits progression. This project will (1) characterize glutamine-dependent MDS subtypes; (2) determine mechanisms and investigate pre-clinical efficacy of the novel, potent, orally bioavailable GLS inhibitor CB-839 in MDS models; and (3) test therapeutic efficacy of CB-839 combined with 5-azacytidine in a Phase I/II clinical trial in patients with intermediate and high-risk MDS. We will specifically determine the efficacy of this combination in eliminating MDS stem cells, and analyze the effects on their methylation states. Since hypermethylation of DNA is an important hallmark of MDS, a combination of two hypomethylating approaches (via distinct mechanisms of action) will be potentially efficacious in MDS. The proposed work will generate a better understanding of the metabolic pathways in MDS.